Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Spin Dynamics of Flavoproteins.

This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field.

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase.

Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F- ) and chloride (Cl- ). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl- and F- bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl- binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F- binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity.

Discovery of Two Novel Oxidases Using a High-Throughput Activity Screen.

Discovery of novel enzymes is a challenging task, yet a crucial one, due to their increasing relevance as chemical catalysts and biotechnological tools. In our work we present a high-throughput screening approach to discovering novel activities. A screen of 96 putative oxidases with 23 substrates led to the discovery of two new enzymes. The first enzyme, N-acetyl-D-hexosamine oxidase (EC 1.1.3.29) from Ralstonia solanacearum, is a vanillyl alcohol oxidase-like flavoprotein displaying the highest activity with N-acetylglucosamine and N-acetylgalactosamine. Before our discovery of the enzyme, its activity was an orphan one - experimentally characterized but lacking the link to amino acid sequence. The second enzyme, from an uncultured marine euryarchaeota, is a long-chain alcohol oxidase (LCAO, EC 1.1.3.20) active with a range of fatty alcohols, with 1-dodecanol being the preferred substrate. The enzyme displays no sequence similarity to previously characterised LCAOs, and thus is a completely novel representative of a protein with such activity.

Dynamic association of flavin cofactors to regulate flavoprotein function.

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.

Optical Redox Imaging Is Responsive to TGFß Receptor Signalling in Triple-Negative Breast Cancer Cells.

Co-enzyme nicotinamide adenine dinucleotide NAD(H) regulates hundreds of biochemical reactions within the cell. We previously reported that NAD(H) redox status may have prognostic value for predicting breast cancer metastasis. However, the mechanisms of NAD(H) involvement in metastasis remain elusive. Given the important roles of TGFß signalling in metastatic processes, such as promoting the epithelial-to-mesenchymal transition, we aimed to investigate the involvement of the mitochondrial NAD(H) redox status in TGFß receptor signalling. Here we present the initial evidence that NAD(H) redox status is responsive to TGFß receptor signalling in triple-negative breast cancer cells in culture. The mitochondrial NAD(H) redox status was determined by the optical redox imaging (ORI) technique. Cultured HCC1806 (less aggressive) and MDA-MB-231 (more aggressive) cells were subjected to ORI after treatment with exogenous TGFß1 or LY2109761, which stimulates or inhibits TGFß receptor signalling, respectively. Cell migration was determined with the transwell migration assay. Global averaging quantification of the ORI images showed that 1) TGFß1 stimulation resulted in differential responses between HCC1806 and MDA-MB-231 lines, with HCC1806 cells having a significant change in the mitochondrial redox status, corresponding to a larger increase in cell migration; 2) HCC1806 cells acutely treated with LY2109761 yielded immediate increases in ORI signals. These preliminary data are the first evidence that suggests the existence of a cell line-dependent shift of the mitochondrial NAD(H) redox status in the TGFß receptor signalling induced migratory process of breast cancer cells. Further research should be conducted to confirm these results as improved understanding of the underlying mechanisms of metastatic process may contribute to the identification of prognostic biomarkers and therapeutic targets.

Specialization in a Nitrogen-Fixing Symbiosis: Proteome Differences Between Sinorhizobium medicae Bacteria and Bacteroids.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

Ferroptosis-Related Flavoproteins: Their Function and Stability.

Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of diverse diseases. From the other side, the induction of ferroptosis can be used to kill tumor cells as a novel therapeutic approach. Because of the broad clinical relevance, a comprehensive understanding of the ferroptosis-controlling protein network is necessary. Noteworthy, several proteins from this network are flavoenzymes. This review is an attempt to present the ferroptosis-related flavoproteins in light of their involvement in anti-ferroptotic and pro-ferroptotic roles. When available, the data on the structural stability of mutants and cofactor-free apoenzymes are discussed. The stability of the flavoproteins could be an important component of the cellular death processes.

Optical Redox Imaging of Treatment Responses to Nampt Inhibition and Combination Therapy in Triple-Negative Breast Cancer Cells.

We evaluated the utility of optical redox imaging (ORI) to identify the therapeutic response of triple-negative breast cancers (TNBC) under various drug treatments. Cultured HCC1806 and MDA-MB-231 cells treated with FK866 (nicotinamide phosphoribosyltransferase (Nampt) inhibitor), FX11 (lactate dehydrogenase A inhibitor), paclitaxel, and their combinations were subjected to ORI, followed by imaging fluorescently labeled reactive oxygen species (ROS). Cell growth inhibition was measured by a cell viability assay. We found that both cell lines experienced significant NADH decrease and redox ratio (Fp/(NADH+Fp)) increase due to FK866 treatment; however, HCC1806 was much more responsive than MDA-MB-231. We further studied HCC1806 with the main findings: (i) nicotinamide riboside (NR) partially restored NADH in FK866-treated cells; (ii) FX11 induced an over 3-fold NADH increase in FK866 or FK866+NR pretreated cells; (iii) FK866 combined with paclitaxel caused synergistic increases in both Fp and the redox ratio; (iv) FK866 sensitized cells to paclitaxel treatments, which agrees with the redox changes detected by ORI; (v) Fp and the redox ratio positively correlated with cell growth inhibition; and (vi) Fp and NADH positively correlated with ROS level. Our study supports the utility of ORI for detecting the treatment responses of TNBC to Nampt inhibition and the sensitization effects on standard chemotherapeutics.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

Flavin-dependent N-hydroxylating enzymes: distribution and application.

Amino groups derived from naturally abundant amino acids or (di)amines can be used as "shuttles" in nature for oxygen transfer to provide intermediates or products comprising N-O functional groups such as N-hydroxy, oxazine, isoxazolidine, nitro, nitrone, oxime, C-, S-, or N-nitroso, and azoxy units. To this end, molecular oxygen is activated by flavin, heme, or metal cofactor-containing enzymes and transferred to initially obtain N-hydroxy compounds, which can be further functionalized. In this review, we focus on flavin-dependent N-hydroxylating enzymes, which play a major role in the production of secondary metabolites, such as siderophores or antimicrobial agents. Flavoprotein monooxygenases of higher organisms (among others, in humans) can interact with nitrogen-bearing secondary metabolites or are relevant with respect to detoxification metabolism and are thus of importance to understand potential medical applications. Many enzymes that catalyze N-hydroxylation reactions have specific substrate scopes and others are rather relaxed. The subsequent conversion towards various N-O or N-N comprising molecules is also described. Overall, flavin-dependent N-hydroxylating enzymes can accept amines, diamines, amino acids, amino sugars, and amino aromatic compounds and thus provide access to versatile families of compounds containing the N-O motif. Natural roles as well as synthetic applications are highlighted. Key points • N-O and N-N comprising natural and (semi)synthetic products are highlighted. • Flavin-based NMOs with respect to mechanism, structure, and phylogeny are reviewed. • Applications in natural product formation and synthetic approaches are provided. Graphical abstract .

Retbindin: A riboflavin Binding Protein, Is Critical for Photoreceptor Homeostasis and Survival in Models of Retinal Degeneration.

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models; one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn-/- and Prph2Y141C/+/Rtbdn-/- retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

Functional and topological diversity of LOV domain photoreceptors.

Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its ß-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme.

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins.

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting PtIV and RuII complexes into potentially toxic PtII or RuII -OH2 species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate PtIV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes PtIV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of PtIV chemotherapeutic agents.

The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions.

Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B2-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions.

Structure of dihydromethanopterin reductase, a cubic protein cage for redox transfer.

Dihydromethanopterin reductase (Dmr) is a redox enzyme that plays a key role in generating tetrahydromethanopterin (H4MPT) for use in one-carbon metabolism by archaea and some bacteria. DmrB is a bacterial enzyme understood to reduce dihydromethanopterin (H2MPT) to H4MPT using flavins as the source of reducing equivalents, but the mechanistic details have not been elucidated previously. Here we report the crystal structure of DmrB from Burkholderia xenovorans at a resolution of 1.9 Å. Unexpectedly, the biological unit is a 24-mer composed of eight homotrimers located at the corners of a cubic cage-like structure. Within a homotrimer, each monomer-monomer interface exhibits an active site with two adjacently bound flavin mononucleotide (FMN) ligands, one deeply buried and tightly bound and one more peripheral, for a total of 48 ligands in the biological unit. Computational docking suggested that the peripheral site could bind either the observed FMN (the electron donor for the overall reaction) or the pterin, H2MPT (the electron acceptor for the overall reaction), in configurations ideal for electron transfer to and from the tightly bound FMN. On this basis, we propose that DmrB uses a ping-pong mechanism to transfer reducing equivalents from FMN to the pterin substrate. Sequence comparisons suggested that the catalytic mechanism is conserved among the bacterial homologs of DmrB and partially conserved in archaeal homologs, where an alternate electron donor is likely used. In addition to the mechanistic revelations, the structure of DmrB could help guide the development of anti-obesity drugs based on modification of the ecology of the human gut.